Analyze particles with ROI manager in stacks

Dear all,

I’m trying to analyze the porosity micro-structure of my bone samples, using Analyze Particles function. When binarized, the structure actually looks very similar to irregularly-shaped cells, which makes it a useful function to measure basic parameters such as the cross-section area (average size), thickness (Feret), and the porosity ratio (Area%) of the bone.

The problem is that the ROI is changing form slide to slide. Using the freehand selection tool, I can draw my desired ROI on each slide, and with the aid of “Interpolate ROI” function, I have my final ROI on each slide. However, it seems that within the ROI manager window, I can only use the basic “measure” function for the selected subset of ROIs. When I use the Analyze particle function, it ignores the created subset, and chooses one fixed ROI for all the stacks! I wonder if I’m missing something? Probably something basic, but I’m not sure what it is.
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ROI%20Manager

Hi Shahed,

I think there is a misunderstanding on what Analyze Particles does. First it segments your binary image i.e. it creates ROIs for each connected region, and then it allows you to measure all these ROIs.

From what I understand what you want is simply to create manually a series of large ROIs that encompass the entire bone on each slide and then measure the properties of those large region. For that you simply create the ROIs as you did, then click on “Deselect” in the ROI manager, and then click on “Measure”. This will measure properties of your ROIs separately for each slide. You can add properties you want to measure (e.g. %Area or Feret’s diameter) in the menu Analyze -> Set Measurements, and they will appear in your results window (you can also select “Stack position” so that you know which measurement corresponds to which slide).

I hope that helps!

Guillaume

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Guillaume
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Hi Guillaume,

Many thanks for the reply.
Maybe I should have been more specific. The structures that I’m interested in are the black areas (porosity) within the predominantly white region (white). When I select “Measure” in the ROI Manager, it wouldn’t only give me the properties of the black areas, but rather an average of the whole structure combined. That’s why I want to use the “analyze Particles” function, to basically trick the software into believing that the black regions are particles (or cells).

Actually, I figure out what the problem was by reading the ImageJ user guide. What I should have done was to go to Image -> Overlay -> From ROI Manager to copy my subset into the original image. Then, I can choose any sort of measurement that I want.

Now there’s another problem. I have about 640 images in one stack, but I want to perform the measurement for 40-50 of the middle slides. How can I specifically define the range of the slides that I want to measure?

Kindest regards,
Shahed

Hi Shahed,

Now I am even more confused by what you are trying to achieve. Let me try to formulate a few questions so that I can better understand and help you:

  1. How do you want to use the manually traced ROIs that you show in the image ?
  2. Analyze particles can indeed create ROIs for all the small black regions. But then “Measure” will measure all these small regions, and parameters like %Area won’t have any meaning in that case (it’s just going to be 100%). You will end up with a long list of parameters for each small region in each slide. How do you want to use that information then ?
  3. Overlays are not things that you measure. “Measure” only measures the ROIs that are in the ROI manager. So I’m not sure what you are trying to achieve here.

Guillaume

Hi Guillaume,

I definitely needed to elaborate on my problem, as well as what I’m trying to achieve.
I’ll explain from scratch again.

There are porous structure in bone, which look like this in the microCT images:

After the binarization, they look like this:

The predominantly black region in the left is “air”, and that’s the region I want to exclude by manual contouring. So, the progression of my ROIs will look like this:

2- The advantage of using “Analyze Particle” function is this:

  • %Area would be the ratio of porous structure (black) to bone (white), in my cumulative ROIs, which is indeed very useful for me (it’s about 5-7% actually)
  • I will have information of the diameter of each porous structure, which can answer critical questions in my thesis.

I hope this has clarified what exactly I’m looking for. Having said this, I have a problem regarding this method that I cannot seem to overcome. When I create my ROIs in the ROI manager, and then click “Analyze Particle”, the outline images indicate that the ROIs are not “moving” as I have drawn them, but rather one single ROI is chosen for all of them:

for the example, in the above slice, I have selected a complete circle in the ROI manager, but in the image, the ROI for the first slice is measured instead. In other words, it seems that not all the ROIs that I’ve drawn in the ROI manager are being used, but only the first one. I know it seems a bit complicated, but I tried to explain it to the best of my ability! :slight_smile:

do you have an idea what I am missing here?